BR112015011036B1 - Heterocyclic Substituted 2-Amino-quinazoline Derivatives, Pharmaceutical Composition - Google Patents

Abstract

HETEROCYCLIC SUBSTITUTED 2-AMINO-QUINAZOLINE DERIVATIVES FOR THE TREATMENT OF VIRAL INFECTIONS. This invention relates to heterocyclic substituted 2-amino-quinazoline derivatives, processes for their preparation, pharmaceutical compositions, and their use in the treatment of viral infections.

Description

[001] This invention relates to heterocyclic substituted 2-amino-quinazoline derivatives, processes for their preparation, pharmaceutical compositions, and their use in the treatment of viral infections.

[002] The present invention relates to the use of heterocyclic substituted 2-amino-quinazoline derivatives in the treatment of viral infections, immune or inflammatory disorders, where modulation, or agonism, of toll-like receptors (TLRs) is involved. Toll-Type Receptors are primary transmembrane proteins characterized by a leucine-rich extracellular domain and a cytoplasmic extension that contains a conserved region. The innate immune system is able to recognize molecular patterns associated with pathogens through these TLRs expressed on the cell surface of certain types of immune cells. Recognition of foreign pathogens activates cytokine production and an up-regulation of costimulatory molecules in phagocytes. This leads to modulation of the behavior of T lymphocytes.

[003] It has been estimated that most mammalian species have between ten and fifteen types of Toll-like receptors. Thirteen TLRs (termed TLR1 to TLR13) have been identified in humans and mice together, and equivalent forms of many of these have been found in other mammalian species. However, equivalents of certain TLRs found in humans are not present in all mammals. For example, a gene encoding a protein analogous to TLR10 in humans is present in mice, but appears to have been damaged at some point in the past by a retrovirus. On the other hand, mice express TLRs 11, 12 and 13, none of which are represented in humans. Other mammals can express TLRs that are not found in humans. Other non-mammalian species may have TLRs distinct from mammals, as demonstrated by TLR14, which is found in the Takifugu pufferfish. This can complicate the process of using experimental animals as models of human innate immunity.

[004] For reviews of TLRs see the following journal articles. Hoffmann, J.A., Nature, 426, pp. 33-38, 2003; Akira, S., Takeda, K., and Kaisho, T., Annual Rev. Immunology, 21, pp. 335-376, 2003; Ulevitch, R.J., Nature Reviews: Immunology, 4, pp. 512-520, 2004.

[005] Compounds indicating activity at Toll Type receptors such as purine derivatives in WO 2006/117670, adenine derivatives in WO 98/01448 and WO 99/28321, and pyrimidines in WO 2009/067081 have previously been described.

[006] However, there is a strong need for new Toll-Type receptor modulators having preferential selectivity, higher potency, higher metabolic stability, and an improved safety profile compared to prior art compounds.

ou um seu sal, tautômero(s), formas estereoisomêricas, solvato ou polimorfo farmaceuticamente aceitáveis, em que[007] According to the present invention there is provided a compound of formula (I)

or a pharmaceutically acceptable salt, tautomer(s), stereoisomeric forms, solvate or polymorph thereof, wherein

[008] R1 is any of the following structures:

[009] R2 is hydrogen, -O-(C1-3)alkyl, halogen, (C1-3)alkyl, -O-(C1-3)alkyl-O-(C1-3)alkyl or CH2OH ;

[0010] R3 is hydrogen, -O-(C1-3)-alkyl, halogen, (C1-3)-alkyl, or -C(=O)-R7 where R7 is -O-(C1-3)-alkyl , NH2, NH(CH3), N(CH3)2, N(CH3)alkyl-(C1-3), N(alkyl-(C1-3))2 or pyrrolidine;

[0011] R4 is hydrogen or fluorine;

[0012] R5 is (C1-3)-alkyl, fluoro-C1-3-alkyl or CH2OH;

[0013] R6 is NH2, NH(CH3) or N(CH3)2, (hetero)-anilines optionally substituted by one or more R8, R9, Ri0, Rii or Ri2 or (hetero)-benzylamines optionally substituted by one or more R8 , R9, Ri0, Rii, or Ri2,

[0014] R8, R9, Ri0, Rii and Ri2, which are the same or different, are each independently selected from hydrogen, (C1-3)-alkyl, -O- (C1-3)-alkyl, or halogen

[0015] and

[0016] R13 is hydrogen, (C1-3)-alkyl or (C1-3)-fluoro-alkyl.

[0017] Preferred compounds according to the invention are compounds numbered 12 and 29 as illustrated in Table II.

[0018] The compounds of formula (I) and their pharmaceutically acceptable salts, tautomer(s), stereoisomeric forms, solvate or polymorph have activity as pharmaceuticals, in particular as modulators of Toll-Type Receptor activity (especially TLR7 activity and/or or TLR8).

[0019] In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, tautomer, stereoisomeric form, solvate or polymorph thereof together with one or more pharmaceutically acceptable excipients, diluents or carriers. .

[0020] Furthermore, a compound of formula (I) or a pharmaceutically acceptable salt, tautomer, stereoisomeric form, solvate or polymorph thereof according to the present invention, or a pharmaceutical composition comprising said compound of formula (I) ) or a pharmaceutically acceptable salt, tautomer, stereoisomeric form, solvate or polymorph thereof can be used as a medicine.

[0021] Another aspect of the invention is that a compound of formula (I) or a pharmaceutically acceptable salt, tautomer, stereoisomeric form, solvate or polymorph thereof, or said pharmaceutical composition comprising said compound of formula (I) or a salt thereof, pharmaceutically acceptable tautomer, stereoisomeric form, solvate or polymorph may be used accordingly in the treatment of a disorder in which modulation of TLR7 and/or TLR8 is involved.

[0022] The term "-(C1-3)alkyl" refers to a straight-chain, branched-chain or cyclic saturated aliphatic hydrocarbon containing the specified number of carbon atoms.

[0023] The term "fluoro-alkyl-(C1-3)" refers to a straight-chain, branched-chain or cyclic saturated aliphatic hydrocarbon containing the specified number of carbon atoms where one or more hydrogen atoms have been replaced by a of fluoride.

[0024] The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably to fluorine and chlorine.

[0025] The term "aniline" refers to a compound of the formula C6H5NR13- consisting of a phenyl group attached to an amino group; with "(hetero)-aniline" it is meant that 1-3 nitrogen atoms are present in the aromatic ring, preferably 1 nitrogen atom.

[0026] The term "benzylamine" refers to a compound of the formula C6H5CH2NR13- consisting of a benzyl group, C6H5CH2, attached to an amine functional group; with "(hetero)-benzylamine" is meant that 1-3 nitrogen atoms are present in the aromatic ring, preferably 1 nitrogen atom.

[0027] As used herein, any chemical formula with bonds shown only as solid lines and not as solid minted bonds or perforated minted bonds, or otherwise indicated as having a particular configuration (e.g., R, S) around a or more atoms, contemplates each possible stereoisomer, or mixture of two or more stereoisomers.

[0028] The terms "stereoisomers", "stereoisomeric forms" or "stereochemically isomeric forms" are previously or hereinafter used interchangeably.

[0029] The invention includes all stereoisomers of the compounds of the invention either as a pure stereoisomer or as a mixture of two or more stereoisomers.

[0030] Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture.

[0031] Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e., they are not related as mirror images. If a compound contains a double bond, the substituents can be in the E or Z configuration. If a compound contains an at least disubstituted non-aromatic cyclic group, the substituents can be in the cis or trans configuration.

[0032] Therefore, the invention includes enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof, whenever chemically possible.

[0033] The meaning of all these terms, i.e., enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof is known to the skilled person.

[0034] Absolute setting is specified according to the Cahn-Ingold-Prelog system. The configuration on an asymmetric atom is specified by R or S. Resolved stereoisomers whose absolute configuration is not known may be designated by (+) or (-) depending on the direction in which they rotate plane polarized light. For example, resolved enantiomers whose absolute configuration is not known may be designated by (+) or (-) depending on the direction in which they rotate plane polarized light.

[0035] When a specific stereoisomer is identified, this means that said stereoisomer is substantially free, ie, associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1%, of the other stereoisomers. Thus, when a compound of Formula (I) is for example specified as (R), this means that the compound is substantially free of the (S)-isomer; when a compound of Formula (I) is for example specified as E, this means that the compound is substantially free of the Z-isomer; when a compound of Formula (I) is for example specified as cis, this means that the compound is substantially free of the trans isomer.

[0036] Pharmaceutically acceptable salts of the compounds of formula (I) include acid and base addition salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Suitable base salts are formed from bases which form non-toxic salts.

[0037] The compounds of the invention may also exist in solvated and unsolvated forms. The term "solvate" is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example ethanol.

[0038] The term "polymorph" refers to the ability of the compound of the invention to exist in more than one form or crystal structure.

[0039] The compounds of the present invention can be administered as crystalline or amorphous products. They can be obtained for example as solid buffers, powders, or films, by methods such as precipitation, crystallization, lyophilization, spray drying, or evaporative drying. They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs. They will generally be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient largely depends on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0040] The compounds of the present invention or any subgroup thereof may be formulated into various pharmaceutical forms for purposes of administration. As appropriate compositions there may be cited all compositions usually employed for systemic drug administration. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier can take a wide variety of forms depending on the form. of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, for example, for oral, rectal, or percutaneous administration. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs. , emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. Also included are solid form preparations which can be converted, immediately before use, to liquid forms. In compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate administration to the skin and/or may be useful for preparing the desired compositions. These compositions can be administered in a variety of ways, e.g., as a transdermal patch, as a patch, as an ointment. The compounds of the present invention may also be administered via inhalation or insufflation by means of methods and formulations employed in the art for administration via this route. Thus, in general the compounds of the present invention can be administered to the lungs in the form of a solution, a suspension or a dry powder.

[0041] It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.

[0042] Those of skill in the treatment of infectious diseases will be able to determine the effective amount from the test results presented hereinafter. In general it is contemplated that an effective daily amount would be from 0.01 mg/kg to 50 mg/kg of body weight, more preferably from 0.1 mg/kg to 10 mg/kg of body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form.

[0043] The exact dosage and frequency of administration depend upon the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient as well as other medication that the individual may be taking it, as is well known to those skilled in the art. Furthermore, it is evident that the effective amount may be decreased or increased depending on the response of the subject treated and/or depending on the judgment of the physician prescribing the compounds of the present invention. The ranges of effective amounts mentioned above are therefore guidelines only and are not intended to limit the scope or use of the invention to any extent. Preparation of compounds of formula (I)

Seção Experimental.Procedimento geral de fabricação de uma 2-amino-4-hidroxiquinazolina substituída

[0044] Compounds of formula (I) are prepared according to scheme 1. Esters or substituted anthranilic acids (II) were heated under acidic conditions in the presence of excess cyanamide, using an alcoholic solvent (e.g. ethanol) or diglyme according to the method described in the literature (O'Hara et al. JOC (1991) 56, p776). Subsequent amine substitution of 2-amino-4-hydroxyquinazolines (III) can proceed via a coupling agent such as BOP or PyBOP in the presence of DBU and the amine in polar aprotic solvent (eg, DMF). 1:

Experimental Section. General procedure for manufacturing a substituted 2-amino-4-hydroxyquinazoline

[0045] In a 500 mL pressure vessel equipped with a magnetic stir bar were placed 2-amino-5-methoxybenzoic acid (25 g, 149.6 mmol, ethanol (200 mL), cyanamide (9.43 g, 224 mmol), and concentrated HCl (6 mL). The mixture was allowed to stir at 100 °C for 16 h. The reaction mixture was allowed to cool to room temperature and the solids were isolated by filtration and washed with ethanol and DIPE The crude product was dried under vacuum at 50°C to obtain an off-white solid.

[0046] LC-MS m/z = 192(M+H)

Procedimento geral de fabricação do composto IV[0047] 1H NMR (400MHz, DMSO-d6) δ ppm 3.88 (s, 3H), 6.96 (dd,J=8.2, 3.1Hz, 2H), 7.69 (t, J=8.3 Hz, 1H), 8.28 (l.s., 2H), 12.67 (l.s., 1H) Table I: Compounds of formula (III). The following intermediates were prepared according to the method for preparing III-1.

General manufacturing procedure for compound IV

[0048] Compound III (1.5 mmol) and DBU (3.75 mmol) were dissolved in 5 mL of DMF in a 30 mL glass vial. After 5 minutes BOP (1.5 mmol) was added. The reaction mixture was stirred for 5 minutes and then the amine (2.25 mmol) was added. The reaction mixture was stirred overnight. The crude reaction mixture was purified by prep HPLC. in (RP Vydac Denali C18 - 10 µm, 250 g, 5 cm). Mobile phase (0.25% solution of NH4HCO3 in water, MeOH), the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain the product as a solid. General procedure for manufacturing compounds 22, 23, 24, 26, 27 and 28

[0049] Compound 8 of formula (I) (see table II) (2.1 g, 6.5 mmol) was dispensed into THF (50 mL), LiOH (409 mg, 9.74 mmol) was added followed by MeOH (5 mL). The reaction mixture was stirred overnight at room temperature. The solvents were evaporated until only water remained. 10 mL of 1M HCl was added and the compound was extracted with 2-methyltetrahydrofuran (2 X 25 mL). The combined organic layers were dried over MgSO4 and the solvents removed under reduced pressure to obtain 2-amino-4-[1-(2-pyridyl)ethylamino]quinazoline-7-carboxylic acid as a white solid.

[0050] 2-Amino-4-[1-(2-pyridyl)ethylamino]quinazoline-7-carboxylic acid (200mg, 0.65mmol) and PyBOP (421mg, 0.81mmol) were dissolved in DMF (5 mL) in a 30 mL glass bottle. After 5 minutes Hunig's base (0.557 mL, 3.23 mmol) was added. The reaction mixture was stirred for 5 minutes and then the amine was added. The reaction mixture was stirred overnight. The crude reaction mixture was purified by prep HPLC. in (RP Vydac Denali C18 - 10 µm, 250 g, 5 cm). Mobile phase (0.25% solution of NH4HCO3 in water, MeOH), the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain the product as a solid.Procedure for making compound 29

[0051] Compound 12 of formula (I) (see table II) (1500mg, 4.78mmol) and pyridine hydrochloride (3.32g, 28.7mmol) were dissolved in pyridine (20ml) and heated to 120°C for 16 h. Pyridine was removed under reduced pressure. The residual fraction was quenched with a solution (sat., aq.) of NaHCO3. The precipitate was filtered, washed with water and dried under vacuum at 50 °C to give a brown solid which was purified by preparative HPLC (Stationary phase: RP Vydac Denali C18 - 10 μm, 200 g, 5 cm), Mobile phase: solution to 0.25% NH4HCO3 in water, CH3CN, the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain 2-amino-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5 -ol (100 mg) as a solid.

[0052] 2-Amino-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5-ol (40mg, 0.15mmol) and Cs2CO3 (144mg, 0.44mmol) were dissolved in DMF (7.5 mL) and stirred at room temperature for 30 minutes. 2-Bromoethyl methyl ether (0.018 mL, 0.18 mmol) was added and the entire mixture was stirred for 16 hours at room temperature. The solvent was removed under reduced pressure and the crude residue was neutralized with 1M HCl and purified by preparative HPLC on (RP Vydac Denali C18 - 10 µm, 250 g, 5 cm). Mobile phase (0.25% solution of NH4HCO3 in water, MeOH), the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain compound 29 as a solid.Procedure for making compound 30

[0053] A 75 mL stainless steel autoclave was charged under N 2 atmosphere with 2-amino-5-bromo-quinazolin-4-ol (3 g, 12.5 mmol), Pd(OAc) 2 (56 mg, 0 .25 mmol), 1,3-bis(diphenylphosphino)propane (206 mg, 0.5 mmol), potassium acetate (2.45 g, 25 mmol), methanol (25 mL) and THF (30 mL). The autoclave was closed and pressurized to CO gas at 50 bar and the reaction was carried out for 16 hours at 100°C. The formed precipitate was filtered off yielding methyl 2-amino-4-hydroxy-quinazoline-5-carboxylate (2.35 g).

[0054] Methyl 2-amino-4-hydroxy-quinazoline-5-carboxylate (2.35 g) in THF (10 mL) was cooled to 0 °C. Then LiAlH4 was added. The mixture was allowed to reach room temperature and stirred for 16 hours. EtOAc (5 mL) was added dropwise at 0°C, then 3 g of Na2SO4.10H2O was added and the whole mixture was stirred for 30 minutes. The precipitate was filtered, and the filtrate was dried over MgSO4 , filtered and evaporated to dryness to obtain 2-amino-5-(hydroxymethyl)quinazolin-4-ol (750 mg) as a yellow solid.

[0055] 2-Amino-5-(hydroxymethyl)quinazolin-4-ol (300mg, 1.57mmol) was suspended in THF (20mL) with DBU (0.586mL, 3.92mmol), after 5 minutes it was BOP (833 mg, 1.88 mmol) is added. After 15 minutes (5-methyl-3-isoxazolyl)methylamine (0.320 mL, 3.14 mmol) was added. The mixture was stirred for 16 hours at room temperature. The solvent was removed under reduced pressure and the crude product was purified by preparative HPLC on (RP Vydac Denali C18 - 10 µm, 250 g, 5 cm). Mobile phase (0.25% solution of NH4HCO3 in water, MeOH), the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain compound 30 as a solid (119 mg). 31

[0056] A freshly prepared solution of NaOMe (1.25 mL, 6.25mmol) was added under N2 atmosphere to a mixture of 2-amino-5-bromo-8-fluoro-quinazolin-4-ol (500 mg , 1.94 mmol), copper(I) bromide (39 mg, 0.27 mmol), EtOAc (0.076 mL, 0.78 mmol) in MeOH (5 mL). The mixture was heated in a pressure vessel to reflux for 16 hours. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (Stationary phase: RP Vydac Denali C18 - 10 μm, 200 g, 5 cm), Mobile phase: 0.25% solution of NH4HCO3 in water, MeOH, the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to give 2-amino-8-fluoro-5-methoxy-quinazolin-4-ol (150 mg) as a solid.

[0057] 2-Amino-8-fluoro-5-methoxy-quinazolin-4-ol (150mg, 0.72mmol) was dispensed into DMF (10mL), DBU (0.536mL, 3.59mmol) was added. and then BOP reagent (396 mg, 0.90 mmol) was added. The reaction mixture was stirred and, when homogeneous, (5-methyl-3-isoxazolyl)methylamine (0.115 mL, 1.08 mmol) was added. The reaction mixture was stirred 16 hours. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC (Stationary phase: RP Vydac Denali C18 - 10 μm, 200 g, 5 cm), Mobile phase: 0.25% solution of NH4HCO3 in water, MeOH, the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain compound 31 as a solid (64 mg).Procedure for making compound 32

[0058] Compound 31 (52.5 mg, 0.173 mmol) and pyridine hydrochloride (0.12 g, 1.039 mmol) in 1 mL of pyridine were heated to 120 °C for 16 hours. Volatiles were removed under reduced pressure. The residue was quenched with a solution (sat., aq.) of NaHCO 3 . The precipitate was filtered, washed with water and dried under vacuum at 50°C to give 2-amino-8-fluoro-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5-ol (10mg) as a brown solid.

[0059] 2-Amino-8-fluoro-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5-ol (10mg, 0.035mmol) and Cs2CO3 (33.8mg, 0.104mmol) in DMF (5 mL) was stirred at room temperature for 30 minutes. 2-Chloroethyl methyl ether (4.1 mg, 0.043 mmol) was added and the entire mixture was stirred for 16 hours at room temperature. The solvent was removed under reduced pressure. The residue was dissolved in MeOH and the precipitate (salts) was filtered off. The filtrate was concentrated under reduced pressure and the crude residue was purified by preparative HPLC on (Stationary phase: RP SunFire Prep C18 OBD-10 μm, 30 x 150 mm), Mobile phase: 0.25% solution of NH4HCO3 in water , CH3CN, the desired fractions were collected, evaporated, dissolved in MeOH and evaporated again to obtain compound 32 as a solid (2mg).Table II: Compounds of formula (I). The following compounds were synthesized according to one of the methods described above.

SFC purification methods. General procedure

[0060] Supercritical Fluid Chromatography (SFC) separation was performed with supercritical CO2 and a modifier as specified in the table using a column as specified in the table. Table III: Compounds of formula (I). The following compounds were isolated by SFC separation.

[0061] For all compounds, the first compound to elute was designated as *R.

[0062] *R means a pure enantiomeric configuration of which the absolute stereochemistry is unknown.

Analytical Methods general procedure

[0063] Measurement by High Performance Liquid Chromatography (HPLC) was performed using an LC pump, a diode array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below).

[0064] The column flow was conducted to the Mass Spectrometer (MS) which was configured with an ion source at atmospheric pressure. It is within the skill of the skilled person to adjust the tuning parameters (eg scan range, residence time...) in order to obtain ions allowing identification of the nominal monoisotropic molecular weight (MW) of the compound. Data acquisition was performed with appropriate software.

[0065] Compounds are described by their experimental retention times (Rt) and ions. If not specified differently in the data table, the reported molecular ion corresponds to [M+H]+ (protonated molecule) and/or [M-H]- (deprotonated molecule). In case the compound is not directly ionizable, the type of adduct is specified (i.e., [M+NH4]+, [M+HCOO]-, etc...). For molecules with multiple isotopic patterns (Br, Cl..), the reported value is that obtained for the lowest isotope mass. All results were obtained with experimental uncertainties that are commonly associated with the method used.

Tabela V: Compostos da fórmula (I). Os seguintes compostos foram caracterizados de acordo com um dos métodos descritos acima.

Atividade Biológica de compostos da fórmula (I)[0066] Hereinafter, "SQD" means Single Quadropole Detector,"MSD" Selective Mass Detector, "TA" room temperature, "BEH" ethylsiloxane/silica bridged hybrid, "DAD" Diode Array Detector, "HSS "High Strength silica. Table IV: LCMS Method Codes (Flow expressed in mL/min; column temperature (T) in °C; Time of

Table V: Compounds of formula (I). The following compounds were characterized according to one of the methods described above.

Biological Activity of compounds of formula (I)

Description of Biological Assays Assessment of TLR7 and TLR8 activity

[0067] The ability of compounds to activate human TLR7 and/or TLR8 was evaluated in a cellular reporter assay using HEK293 cells transiently transfected with a TLR7 or TLR8 expression vector and NFKB-IUC reporter construct.

[0068] Briefly, HEK293 cells were grown in culture medium (DMEM supplemented with 10% FCS and 2mM Glutamine). For transfection of cells in 15 cm dishes, cells were detached with Trypsin-EDTA, transfected with a mixture of plasmid CMV-TLR7 or TLR8 (1700 ng), plasmid NFKB-IUC (850 ng) and a transfection reagent and incubated. for 48 h at 37 °C in a humidified 5 % CO2 atmosphere. The transfected cells were then washed in PBS, detached with Trypsin-EDTA and resuspended in medium to a density of 1.25 x 10 5 cells/ml. Forty microliters of cells were then dispensed into each well in 384-well plates, where 200 nL of compound in 100% DMSO was already present. After 6 hours incubation at 37 °C, 5% CO2, luciferase activity was determined by adding 15 μL of Steady Lite Plus substrate (Perkin Elmer) to each well and reading performed on a ViewLux ultraHTS microplate imager ( Perkin Elmer). Dose response curves were generated from measurements performed in quadruplicate. Minimum effective concentration (LEC) values, defined as the concentration that induces an effect that is at least twice the standard deviation of the assay, were determined for each compound.

[0069] Compound toxicity was determined in parallel using a similar dilution series of compound at 40 µL per well of cells transfected with the CMV-TLR7 construct alone (1.25 x 10 5 cells/mL), in 384-well plates. . Cell viability was measured after 6 hours incubation at 37°C, 5% CO2, by adding 15 μL ATP lite (Perkin Elmer) per well and reading on a ViewLux ultraHTS microplate imager (Perkin Elmer). Data were reported as CC50.

[0070] In parallel, a similar dilution series of compound was used (200 nL of compound in 100% DMSO) with 40 µL per well of cells transfected with the NFKB-IUC reporter construct (1.25 x 10 5 cells/mL) . Six hours after incubation at 37 °C, 5% CO2, luciferase activity was determined by adding 15 μL of Steady Lite Plus substrate (Perkin Elmer) to each well and reading performed on a ViewLux ultraHTS microplate imager (Perkin Elmer). Counter tracking data is reported as LEC.

Activation of ISRE promoter elements

[0071] The potential of compounds to induce IFN-I was also evaluated by measuring the activation of interferon-stimulated responsive elements (ISRE) by PBMC conditioned media. The ISRE element of the GAAACTGAAACT sequence is highly responsive to the transcription factor STAT1-STAT2-IRF9, activated upon binding of IFN-I to its IFNAR receptor (Clontech, PT3372-5W). The pISRE-Luc plasmid from Clontech (ref. 631913) contains 5 copies of this ISRE element, followed by the firefly luciferase ORF. A HEK293 cell line stably transfected with pISRE-Luc (HEK-ISREluc) was established to characterize the conditioned PBMC cell culture medium.

NA = não disponível. Todos os compostos não mostraram nenhuma toxicidade até a concentrada testada mais elevada. Todos os compostos não mostraram nenhuma atividade (LEC >25 μM) no ensaio de contrarrastreio HEK 293 NF-kB descrito acima.[0072] Briefly, PBMCs were prepared from buffy coats of at least two donors using a standard Ficoll centrifugation protocol. Isolated PBMCs were resuspended in RPMI medium supplemented with 10% human AB serum and 2 x 10 5 cells/well were dispensed into 384-well plates containing compounds (total volume 70 µL). After overnight incubation, 10 µL of supernatant was transferred to 384-well plates containing 5 x 10 3 HEK-ISREluc cells/well in 30 µL (plated the day before). After 24 hours of incubation, activation of ISRE elements was measured by assessing luciferase activity using 40 μL/well of Steady Lite Plus substrate (Perkin Elmer) and measured with a ViewLux ultraHTS microplate imager (Perkin Elmer). The stimulatory activity of each compound on HEK-ISREluc cells was reported as the LEC value, defined as the concentration of compound applied to the PBMCs resulting in a luciferase activity at least twice the standard deviation of the assay. The LEC in turn indicates the degree of ISRE activation after transfer of a defined amount of PBMC culture medium. Recombinant interferon α-2a (Roferon-A) was used as a standard control compound.TABLE VI: BIOLOGICAL ACTIVITY.

NA = not available. All compounds showed no toxicity up to the highest tested concentrate. All compounds showed no activity (LEC >25 μM) in the HEK 293 NF-kB counterscreen assay described above.

Claims (5)

1. Compound, characterized by the fact that it has the formula (I)

or a pharmaceutically acceptable salt or tautomer(s) thereof, wherein R1 is any of the following structures:

R2 is hydrogen, -O-(C1-3)alkyl, halogen, (C1-3)alkyl, -O-(C1-3)alkyl-O-(C1-3)alkyl or CH2OH;R3 is hydrogen, -O-(C1-3)-alkyl, halogen, (C1-3)-alkyl, or -C(=O)-R7 where R7 is -O-(C1-3)-alkyl, NH2, NH( CH3), N(CH3)2, N(CH3)-(C1-3)alkyl, N((C1-3)alkyl)2 or pyrrolidine;R4 is hydrogen or fluorine;R5 is (C1-3)alkyl or (hetero)-benzylamines optionally substituted by one or more R8, R9, Ri0, Rii, or Ri2,R8, R9, Ri0, Rii and Ri2, which are the same or different, are each independently selected from hydrogen, alkyl-( C1-3), -O-C1-3 alkyl or halogen, and R13 is hydrogen, C1-3 alkyl or fluoro-C1-3 alkyl. 2. Pharmaceutical composition, characterized in that it comprises a compound of formula (I) or a pharmaceutically acceptable salt or tautomer(s) thereof as defined in claim i, together with one or more pharmaceutically acceptable excipients, diluents or carriers. 3. A compound of formula (I) or a pharmaceutically acceptable salt or tautomer(s) thereof according to claim i, characterized in that it is for use as a medicament. 4. Pharmaceutical composition according to claim 2, characterized in that it is for use as a medicine. 5. A compound of formula (I) according to claim i, characterized in that it is selected from the group consisting of: